Methods of treating hypertriglyceridemia

ABSTRACT

In various embodiments, the present invention provides methods of treating and/or preventing cardiovascular-related disease and, in particular, a method of blood lipid therapy comprising administering to a subject in need thereof a pharmaceutical composition comprising eicosapentaenoic acid or a derivative thereof.

BACKGROUND

Cardiovascular disease is one of the leading causes of death in theUnited States and most European countries. It is estimated that over 70million people in the United States alone suffer from a cardiovasculardisease or disorder including but not limited to high blood pressure,coronary heart disease, dyslipidemia, congestive heart failure andstroke. A need exists for improved treatments for cardiovasculardiseases and disorders.

SUMMARY

In various embodiments, the present invention provides methods oftreating and/or preventing cardiovascular-related diseases and, inparticular, a method of blood lipid therapy comprising administering toa subject in need thereof a pharmaceutical composition comprisingeicosapentaenoic acid or a derivative thereof. In one embodiment, thecomposition contains not more than 10%, by weight, docosahexaenoic acidor derivative thereof, substantially no docosahexaenoic acid orderivative thereof, or no docosahexaenoic acid or derivative thereof. Inanother embodiment, eicosapentaenoic acid ethyl ester comprises at least96%, by weight, of all fatty acids present in the composition; thecomposition contains not more than 4%, by weight, of total fatty acidsother than eicosapentaenoic acid ethyl ester; and/or the compositioncontains about 0.1% to about 0.6% of at least one fatty acid other thaneicosapentaenoic acid ethyl ester and docosahexaenoic acid (orderivative thereof).

In one embodiment, a pharmaceutical composition useful in accordancewith the invention comprises, consists of or consists essentially of atleast 95% by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about0.5% by weight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about0.25% by weight ethyl nonaecapentaenoate (NDPA-E), about 0.2% to about0.45% by weight ethyl arachidonate (AA-E), about 0.3% to about 0.5% byweight ethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32%ethyl heneicosapentaenoate (HPA-E). In another embodiment, thecomposition is present in a capsule shell. In another embodiment, thecomposition contains substantially no or no amount of docosahexaenoicacid (DHA) or derivative thereof such as ethyl-DHA (DHA-E).

In another embodiment, the invention provides a method of treatingmoderate to severe hypertriglyceridemia comprising administering acomposition as described herein to a subject in need thereof one toabout four times per day. In another embodiment, the invention providesa method of treating moderate to severe hypertriglyceridemia comprisingadministering to a subject about 2 g to about 4 g per day of EPA (e.g.,ethyl eicosapentaenoate), for example about 3.8 g to about 4 g per day.

These and other embodiments of the present invention will be disclosedin further detail herein below.

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graphical representation of atherogenic lipoproteinparticle concentration plotted against apolipoprotein B (ApoB) at week12 (N=177; r=0.79).

DETAILED DESCRIPTION

While the present invention is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the invention, and is not intended to limit theinvention to the specific embodiments illustrated. Headings are providedfor convenience only and are not to be construed to limit the inventionin any manner. Embodiments illustrated under any heading may be combinedwith embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specifiedin this application, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited as well as anyranges that can be formed by such values. Also disclosed herein are anyand all ratios (and ranges of any such ratios) that can be formed bydividing a disclosed numeric value into any other disclosed numericvalue. Accordingly, the skilled person will appreciate that many suchratios, ranges, and ranges of ratios can be unambiguously derived fromthe numerical values presented herein and in all instances such ratios,ranges, and ranges of ratios represent various embodiments of thepresent invention.

In one embodiment, the invention provides a method for treatment and/orprevention of a cardiovascular-related disease. The term“cardiovascular-related disease” herein refers to any disease ordisorder of the heart or blood vessels (i.e. arteries and veins) or anysymptom thereof. Non-limiting examples of cardiovascular-related diseaseand disorders include hypertriglyceridemia, hypercholesterolemia, mixeddyslipidemia, coronary heart disease, vascular disease, stroke,atherosclerosis, arrhythmia, hypertension, myocardial infarction, andother cardiovascular events.

The term “treatment” in relation a given disease or disorder, includes,but is not limited to, inhibiting the disease or disorder, for example,arresting the development of the disease or disorder; relieving thedisease or disorder, for example, causing regression of the disease ordisorder; or relieving a condition caused by or resulting from thedisease or disorder, for example, relieving, preventing or treatingsymptoms of the disease or disorder. The term “prevention” in relationto a given disease or disorder means: preventing the onset of diseasedevelopment if none had occurred, preventing the disease or disorderfrom occurring in a subject that may be predisposed to the disorder ordisease but has not yet been diagnosed as having the disorder ordisease, and/or preventing further disease/disorder development ifalready present.

In one embodiment, the present invention provides a method of bloodlipid therapy comprising administering to a subject or subject group inneed thereof a pharmaceutical composition as described herein. Inanother embodiment, the subject or subject group hashypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia and/orvery high triglycerides.

In another embodiment, the subject or subject group being treated has abaseline triglyceride level (or median baseline triglyceride level inthe case of a subject group), fed or fasting, of at least about 300mg/dl, at least about 400 mg/dl, at least about 500 mg/dl, at leastabout 600 mg/dl, at least about 700 mg/dl, at least about 800 mg/dl, atleast about 900 mg/dl, at least about 1000 mg/dl, at least about 1100mg/dl, at least about 1200 mg/dl, at least about 1300 mg/dl, at leastabout 1400 mg/dl, or at least about 1500 mg/dl, for example about 400mg/dl to about 2500 mg/dl, about 450 mg/dl to about 2000 mg/dl or about500 mg/dl to about 1500 mg/dl.

In one embodiment, the subject or subject group being treated inaccordance with methods of the invention has previously been treatedwith Lovaza® and has experienced an increase in, or no decrease in,LDL-C levels and/or non-HDL-C levels. In one such embodiment, Lovaza®therapy is discontinued and replaced by a method of the presentinvention.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of free EPA (or mean thereof in the case of asubject group) not greater than about 0.70 nmol/ml, not greater thanabout 0.65 nmol/ml, not greater than about 0.60 nmol/ml, not greaterthan about 0.55 nmol/ml, not greater than about 0.50 nmol/ml, notgreater than about 0.45 nmol/ml, or not greater than about 0.40 nmol/ml.In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a baseline fastingplasma level (or mean thereof) of free EPA, expressed as a percentage oftotal free fatty acid, of not more than about 3%, not more than about2.5%, not more than about 2%, not more than about 1.5%, not more thanabout 1%, not more than about 0.75%, not more than about 0.5%, not morethan about 0.25%, not more than about 0.2% or not more than about 0.15%.In one such embodiment, free plasma EPA and/or total fatty acid levelsare determined prior to initiating therapy.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of total fatty acid (or mean thereof) not greaterthan about 250 nmol/ml, not greater than about 200 nmol/ml, not greaterthan about 150 nmol/ml, not greater than about 100 nmol/ml, or notgreater than about 50 nmol/ml.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineplasma, serum or red blood cell membrane EPA level not greater thanabout 70 μg/ml, not greater than about 60 μg/ml, not greater than about50 μg/ml, not greater than about 40 μg/ml, not greater than about 30μg/ml, or not greater than about 25 μg/ml.

In another embodiment, methods of the present invention comprise a stepof measuring the subject's (or subject group's mean) baseline lipidprofile prior to initiating therapy. In another embodiment, methods ofthe invention comprise the step of identifying a subject or subjectgroup having one or more of the following: baseline non-HDL-C value ofabout 200 mg/dl to about 400 mg/dl, for example at least about 210mg/dl, at least about 220 mg/dl, at least about 230 mg/dl, at leastabout 240 mg/dl, at least about 250 mg/dl, at least about 260 mg/dl, atleast about 270 mg/dl, at least about 280 mg/dl, at least about 290mg/dl, or at least about 300 mg/dl; baseline total cholesterol value ofabout 250 mg/dl to about 400 mg/dl, for example at least about 260mg/dl, at least about 270 mg/dl, at least about 280 mg/dl or at leastabout 290 mg/dl; baseline vLDL-C value of about 140 mg/dl to about 200mg/dl, for example at least about 150 mg/dl, at least about 160 mg/dl,at least about 170 mg/dl, at least about 180 mg/dl or at least about 190mg/dl; baseline HDL-C value of about 10 to about 60 mg/dl, for examplenot more than about 40 mg/dl, not more than about 35 mg/dl, not morethan about 30 mg/dl, not more than about 25 mg/dl, not more than about20 mg/dl, or not more than about 15 mg/dl; and/or baseline LDL-C valueof about 50 to about 300 mg/dl, for example not less than about 100mg/dl, not less than about 90 mg/dl, not less than about 80 mg/dl, notless than about 70 mg/dl, not less than about 60 mg/dl or not less thanabout 50 mg/dl.

In a related embodiment, upon treatment in accordance with the presentinvention, for example about 2 g to about 4 g per day of a compositionas described herein over a period of about 1 to about 200 weeks, about 1to about 100 weeks, about 1 to about 80 weeks, about 1 to about 50weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1 toabout 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits one or more of the following outcomes:

-   -   (a) reduced triglyceride levels compared to baseline;    -   (b) reduced Apo B levels compared to baseline;    -   (c) increased HDL-C levels compared to baseline;    -   (d) no increase in LDL-C levels compared to baseline;    -   (e) a reduction in LDL-C levels compared to baseline;    -   (f) a reduction in non-HDL-C levels compared to baseline;    -   (g) a reduction in vLDL levels compared to baseline;    -   (h) an increase in apo A-I levels compared to baseline;    -   (i) an increase in apo A-I/apo B ratio compared to baseline;    -   (j) a reduction in lipoprotein A levels compared to baseline;    -   (k) a reduction in LDL particle number compared to baseline;    -   (l) an increase in LDL size compared to baseline;    -   (m) a reduction in remnant-like particle cholesterol compared to        baseline;    -   (n) a reduction in oxidized LDL compared to baseline;    -   (o) no change or a reduction in fasting plasma glucose (FPG)        compared to baseline;    -   (p) a reduction in hemoglobin A_(1c) (HbA_(1c)) compared to        baseline;    -   (q) a reduction in homeostasis model insulin resistance compared        to baseline;    -   (r) a reduction in lipoprotein associated phospholipase A2        compared to baseline;    -   (s) a reduction in intracellular adhesion molecule-1 compared to        baseline;    -   (t) a reduction in interleukin-6 compared to baseline;    -   (u) a reduction in plasminogen activator inhibitor-1 compared to        baseline;    -   (v) a reduction in high sensitivity C-reactive protein (hsCRP)        compared to baseline;    -   (w) an increase in serum or plasma EPA compared to baseline;    -   (x) an increase in red blood cell (RBC) membrane EPA compared to        baseline; and/or    -   (y) a reduction or increase in one or more of serum phospholipid        and/or red blood cell content of docosahexaenoic acid (DHA),        docosapentaenoic acid (DPA), arachidonic acid (AA), palmitic        acid (PA), staeridonic acid (SA) or oleic acid (OA) compared to        baseline.

In one embodiment, upon administering a composition of the invention toa subject, the subject exhibits a decrease in triglyceride levels, anincrease in the concentrations of EPA and DPA (n-3) in red blood cells,and an increase of the ratio of EPA:arachidonic acid in red blood cells.In a related embodiment the subject exhibits substantially no or noincrease in RBC DHA.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(y) above priorto dosing the subject or subject group. In another embodiment, themethods comprise administering a composition as disclosed herein to thesubject after baseline levels of one or more markers set forth in(a)-(y) are determined, and subsequently taking an additionalmeasurement of said one or more markers.

In another embodiment, upon treatment with a composition of the presentinvention, for example about 2 g to about 4 g per day of a compositionas described herein, about 2 g to about 4 g per day of EPA (e.g., ethyleicosapentaenoate), or about 3.8 g to about 4 g per day of EPA (e.g.,ethyl eicosapentaenoate) over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more, any 24 or more, or all 25 of outcomes (a)-(y) describedimmediately above.

In another embodiment, upon treatment with a composition of the presentinvention, the subject or subject group exhibits one or more of thefollowing outcomes:

(a) a reduction in triglyceride level of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) as compared to baseline;

(b) a less than 30% increase, less than 20% increase, less than 10%increase, less than 5% increase or no increase in non-HDL-C levels or areduction in non-HDL-C levels of at least about 1%, at least about 3%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55% or at least about 75% (actual % change or median % change) ascompared to baseline;

(c) substantially no change in HDL-C levels, no change in HDL-C levels,or an increase in HDL-C levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline;

(d) a less than 60% increase, a less than 50% increase, a less than 40%increase, a less than 30% increase, less than 20% increase, less than10% increase, less than 5% increase or no increase in LDL-C levels or areduction in LDL-C levels of at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 55% or at leastabout 75% (actual % change or median % change) as compared to baseline;

(e) a decrease in Apo B levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline;

(f) a reduction in vLDL levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, or at least about 100% (actual % change or median %change) compared to baseline;

(g) a reduction in large (>60 nm e.g. as measured by NMR spectroscopy)vLDL particle concentration, a reduction in large vLDL particleconcentration of at least about 10%, or a reduction in large vLDLparticle concentration of at least about 20% (actual % change or median% change) compared to baseline or placebo control;

(h) a reduction in small (18-20.5 nm; e.g. as measured by NMRspectroscopy) LDL particle concentration, a reduction in small LDLparticle concentration of at least about 3%, a reduction in small LDLparticle concentration of at least about 6%, or a reduction small LDLparticle concentration of at least about 9% (actual % change or median %change) compared to baseline or placebo control;

(i) an increase in apo A-I levels of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline;

(j) an increase in apo A-I/apo B ratio of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline;

(k) a reduction in lipoprotein (a) levels of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline;

(l) a reduction in mean LDL particle number of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline;

(m) an increase in mean LDL particle size of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline;

(n) a reduction in remnant-like particle cholesterol of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline;

(o) a reduction in oxidized LDL of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline;

(p) substantially no change, no significant change, or a reduction (e.g.in the case of a diabetic subject) in fasting plasma glucose (FPG) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline;

(q) substantially no change, no significant change or a reduction inhemoglobin A_(1c) (HbA_(1c)) of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% (actual % change or median % change) compared tobaseline;

(r) a reduction in homeostasis model index insulin resistance of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline;

(s) a reduction in lipoprotein associated phospholipase A2 of at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, or at least about 100%(actual % change or median % change) compared to baseline;

(t) a reduction in intracellular adhesion molecule-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline;

(u) a reduction in interleukin-6 of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline;

(v) a reduction in plasminogen activator inhibitor-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline;

(w) a reduction in high sensitivity C-reactive protein (hsCRP) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline;

(x) an increase in serum, plasma and/or RBC EPA of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 100%, at least about200% or at least about 400% (actual % change or median % change)compared to baseline;

(y) an increase in serum phospholipid and/or red blood cell membrane EPAof at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, r at least about 50%, at leastabout 100%, at least about 200%, or at least about 400% (actual % changeor median % change) compared to baseline;

(z) a reduction or increase in one or more of serum phospholipid and/orred blood cell DHA, DPA, AA, PA and/or OA of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline; and/or

(aa) a reduction in total cholesterol of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(aa) prior todosing the subject or subject group. In another embodiment, the methodscomprise administering a composition as disclosed herein to the subjectafter baseline levels of one or more markers set forth in (a)-(aa) aredetermined, and subsequently taking a second measurement of the one ormore markers as measured at baseline for comparison thereto.

In another embodiment, upon treatment with a composition of the presentinvention, for example about 2 g to about 4 g per day of a compositionas described herein, about 2 g to about 4 g per day of EPA (e.g., ethyleicosapentaenoate), or about 3.8 g to about 4 g per day of EPA (e.g.,ethyl eicosapentaenoate) over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more of, any 24 or more of, any 25 or more of, any 26 or more of,or all 27 or more of outcomes (a)-(aa) described immediately above.

Parameters (a)-(aa) can be measured in accordance with any clinicallyacceptable methodology. For example, triglycerides, total cholesterol,HDL-C and fasting blood sugar can be sample from serum and analyzedusing standard photometry techniques. VLDL-TG, LDL-C and VLDL-C can becalculated or determined using serum lipoprotein fractionation bypreparative ultracentrifugation and subsequent quantitative analysis byrefractometry or by analytic ultracentrifugal methodology. Apo A1, Apo Band hsCRP can be determined from serum using standard nephelometrytechniques. Lipoprotein (a) can be determined from serum using standardturbidimetric immunoassay techniques. LDL particle number and particlesize can be determined using nuclear magnetic resonance (NMR)spectrometry. Remnants lipoproteins and LDL-phospholipase A2 can bedetermined from EDTA plasma or serum and serum, respectively, usingenzymatic immunoseparation techniques. Oxidized LDL, intercellularadhesion molecule-1 and interleukin-6 levels can be determined fromserum using standard enzyme immunoassay techniques. These techniques aredescribed in detail in standard textbooks, for example TietzFundamentals of Clinical Chemistry, 6^(th) Ed. (Burtis, Ashwood andBorter Eds.), WB Saunders Company.

In one embodiment, subjects fast for up to 12 hours prior to bloodsample collection, for example about 10 hours.

In another embodiment, the present invention provides a method oftreating or preventing primary hypercholesterolemia and/or mixeddyslipidemia (Fredrickson Types IIa and IIb) in a patient in needthereof, comprising administering to the patient one or morecompositions as disclosed herein. In a related embodiment, the presentinvention provides a method of reducing triglyceride levels in a subjector subjects when treatment with a statin or niacin extended-releasemonotherapy is considered inadequate (Frederickson type IVhyperlipidemia).

In another embodiment, the present invention provides a method oftreating or preventing risk of recurrent nonfatal myocardial infarctionin a patient with a history of myocardial infarction, comprisingadministering to the patient one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method ofslowing progression of or promoting regression of atheroscleroticdisease in a patient in need thereof, comprising administering to asubject in need thereof one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method oftreating or preventing very high serum triglyceride levels (e.g. TypesIV and V hyperlipidemia) in a patient in need thereof, comprisingadministering to the patient one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method oftreating subjects having very high serum triglyceride levels (e.g.greater than 1000 mg/dl or greater than 2000 mg/dl) and that are at riskof developing pancreatitis, comprising administering to the patient oneor more compositions as disclosed herein.

In another embodiment, the present invention provides a method oftreating subjects having very high serum triglyceride levels (e.g.,greater than 1000 mg/dl or greater than 2000 mg/dl) comprisingadministering to the subject(s) about 2 g to about 4 g per day of EPA(e.g., ethyl eicosapentaenoate), for example about 3.8 g to about 4 gper day of EPA.

In another embodiment, the present invention provides a method ofreducing large vLDL particle concentration in a subject having a fastingbaseline triglyceride level of about 500 mg/dl to about 1500 mg/dl, themethod comprising administering to the subject about 2 g to about 4 gper day, or about 3.8 g to about 4 g per day, of ethyl eicosapentaenoatethereby to produce a reduction in large vLDL particle concentration. Insome embodiments, the reduction is statistically significant. In someembodiments, the reduction in large vLDL particle concentration is incomparison to a baseline large vLDL particle concentration prior toinitiation of administration. In some embodiments, the reduction inlarge vLDL particle concentration is at least a 2.5% reduction, at leasta 10% reduction, at least a 20% reduction, or at least a 24% reduction.In some embodiments, the subject is administered ethyl eicosapentaenoatefor a period of 12 weeks.

In another embodiment, the present invention provides a method ofreducing small LDL particle concentration in a subject having a fastingbaseline triglyceride level of about 500 mg/dl to about 1500 mg/dlcomprising, administering to the subject about 2 g to about 4 g per dayof a pharmaceutical composition comprising at least about 96%, byweight, ethyl eicosapentaenoate thereby to produce a reduction in smallLDL particle concentration by comparison with a baseline small LDLparticle concentration prior to initiation of administration. In someembodiments, the reduction in small LDL particle concentration is atleast a 5% reduction or at least a 9% reduction. In some embodiments,the subject is administered ethyl eicosapentaenoate for a period of 12weeks.

In one embodiment, a composition of the invention is administered to asubject in an amount sufficient to provide a daily dose ofeicosapentaenoic acid of about 2 g to about 4 g, about 3.8 g to about 4g, about 1 mg to about 10,000 mg, 25 about 5000 mg, about 50 to about3000 mg, about 75 mg to about 2500 mg, or about 100 mg to about 1000 mg,for example about 75 mg, about 100 mg, about 125 mg, about 150 mg, about175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about1050 mg, about 1075 mg, about 1100 mg, about 1025 mg, about 1050 mg,about 1075 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500 mg,about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg,about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg,about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about2450 mg, about 2475 mg or about 2500 mg, about 2750 mg, about 3000 mg,about 3250 mg, about 3500 mg, about 3750 mg, about 4000 mg, about 4250mg, about 4500 mg, about 4750 mg, about 5000 mg, about 5500 mg, about6000 mg, about 6500 mg, about 7000 mg, about 7500 mg, about 8000 mg,about 8500 mg, about 9000 mg, about 9500 mg, or about 10,000 mg.

In another embodiment, any of the methods disclosed herein are used intreatment or prevention of a subject or subjects that consume atraditional Western diet. In one embodiment, the methods of theinvention include a step of identifying a subject as a Western dietconsumer or prudent diet consumer and then treating the subject if thesubject is deemed a Western diet consumer. The term “Western diet”herein refers generally to a typical diet consisting of, by percentageof total calories, about 45% to about 50% carbohydrate, about 35% toabout 40% fat, and about 10% to about 15% protein. A Western diet mayalternately or additionally be characterized by relatively high intakesof red and processed meats, sweets, refined grains, and desserts, forexample more than 50%, more than 60% or more or 70% of total caloriescome from these sources.

In one embodiment, a composition for use in methods of the inventioncomprises eicosapentaenoic acid, or a pharmaceutically acceptable ester,derivative, conjugate or salt thereof, or mixtures of any of theforegoing, collectively referred to herein as “EPA.” The term“pharmaceutically acceptable” in the present context means that thesubstance in question does not produce unacceptable toxicity to thesubject or interaction with other components of the composition.

In one embodiment, the EPA comprises all-ciseicosa-5,8,11,14,17-pentaenoic acid. In another embodiment, the EPAcomprises an eicosapentaenoic acid ester. In another embodiment, the EPAcomprises a C₁-C₅ alkyl ester of eicosapentaenoic acid. In anotherembodiment, the EPA comprises eicosapentaenoic acid ethyl ester,eicosapentaenoic acid methyl ester, eicosapentaenoic acid propyl ester,or eicosapentaenoic acid butyl ester. In another embodiment, the EPAcomprises In one embodiment, the EPA comprises all-ciseicosa-5,8,11,14,17-pentaenoic acid ethyl ester.

In another embodiment, the EPA is in the form of ethyl-EPA, lithium EPA,mono-, di- or triglyceride EPA or any other ester or salt of EPA, or thefree acid form of EPA. The EPA may also be in the form of a2-substituted derivative or other derivative which slows down its rateof oxidation but does not otherwise change its biological action to anysubstantial degree.

In another embodiment, EPA is present in a composition useful inaccordance with methods of the invention in an amount of about 50 mg toabout 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about1000 mg, for example about 75 mg, about 100 mg, about 125 mg, about 150mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1025 mg, about1050 mg, about 1075 mg, about 1200 mg, about 1225 mg, about 1250 mg,about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg,about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg,about 1975 mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075mg, about 2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about2200 mg, about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg,about 2325 mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425mg, about 2450 mg, about 2475 mg, about 2500 mg, about 2525 mg, about2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650 mg,about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about 2775mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg, about2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000 mg,about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about 3125mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg, about3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350 mg,about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about 3475mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg, about3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700 mg,about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about 3825mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg, about3950 mg, about 3975 mg, about 4000 mg, about 4025 mg, about 4050 mg,about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about 4175mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg, about4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400 mg,about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about 4525mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg, about4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750 mg,about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about 4875mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, or about5000 mg.

In another embodiment, a composition useful in accordance with theinvention contains not more than about 10%, not more than about 9%, notmore than about 8%, not more than about 7%, not more than about 6%, notmore than about 5%, not more than about 4%, not more than about 3%, notmore than about 2%, not more than about 1%, or not more than about 0.5%,by weight, docosahexaenoic acid (DHA), if any. In another embodiment, acomposition of the invention contains substantially no docosahexaenoicacid. In still another embodiment, a composition useful in the presentinvention contains no docosahexaenoic acid and/or derivative thereof.

In another embodiment, EPA comprises at least 70%, at least 80%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, by weight, of all fatty acids present in acomposition that is useful in methods of the present invention.

In one embodiment, a composition of the invention comprises ultra-pureEPA. The term “ultra-pure” as used herein with respect to EPA refers toa composition comprising at least 95% by weight EPA (as the term “EPA”is defined and exemplified herein). Ultra-pure EPA comprises at least96% by weight EPA, at least 97% by weight EPA, or at least 98% by weightEPA, wherein the EPA is any form of EPA as set forth herein.

In another embodiment, a composition useful in accordance with methodsof the invention contains less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, less than 1%, less than 0.5% or less than 0.25%, by weightof the total composition or by weight of the total fatty acid content,of any fatty acid other than EPA. Illustrative examples of a “fatty acidother than EPA” include linolenic acid (LA), arachidonic acid (AA),docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), stearadonic acid(STA), eicosatrienoic acid (ETA) and/or docosapentaenoic acid (DPA). Inanother embodiment, a composition useful in accordance with methods ofthe invention contains about 0.1% to about 4%, about 0.5% to about 3%,or about 1% to about 2%, by weight, of total fatty acids other than EPAand/or DHA.

In another embodiment, a composition useful in accordance with theinvention has one or more of the following features: (a)eicosapentaenoic acid ethyl ester represents at least about 96%, atleast about 97%, or at least about 98%, by weight, of all fatty acidspresent in the composition; (b) the composition contains not more thanabout 4%, not more than about 3%, or not more than about 2%, by weight,of total fatty acids other than eicosapentaenoic acid ethyl ester; (c)the composition contains not more than about 0.6%, not more than about0.5%, or not more than about 0.4% of any individual fatty acid otherthan eicosapentaenoic acid ethyl ester; (d) the composition has arefractive index (20° C.) of about 1 to about 2, about 1.2 to about 1.8or about 1.4 to about 1.5; (e) the composition has a specific gravity(20° C.) of about 0.8 to about 1.0, about 0.85 to about 0.95 or about0.9 to about 0.92; (e) the composition contains not more than about 20ppm, not more than about 15 ppm or not more than about 10 ppm heavymetals, (f) the composition contains not more than about 5 ppm, not morethan about 4 ppm, not more than about 3 ppm, or not more than about 2ppm arsenic, and/or (g) the composition has a peroxide value of not morethan about 5 meq/kg, not more than about 4 meq/kg, not more than about 3meq/kg, or not more than about 2 meq/kg.

In another embodiment, a composition useful in accordance with theinvention comprises, consists of or consists essentially of at least 95%by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about 0.5% byweight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about 0.25% byweight ethyl nonaecapentaenoate (NDPA-E), about 0.2% to about 0.45% byweight ethyl arachidonate (AA-E), about 0.3% to about 0.5% by weightethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32% ethylheneicosapentaenoate (HPA-E). In another embodiment, the composition ispresent in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 95%,96% or 97%, by weight, ethyl eicosapentaenoate, about 0.2% to about 0.5%by weight ethyl octadecatetraenoate, about 0.05% to about 0.25% byweight ethyl nonaecapentaenoate, about 0.2% to about 0.45% by weightethyl arachidonate, about 0.3% to about 0.5% by weight ethyleicosatetraenoate, and about 0.05% to about 0.32% ethylheneicosapentaenoate. Optionally, the composition contains not more thanabout 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivativethereof such as ethyl-DHA. In one embodiment the composition containssubstantially no or no amount of DHA or derivative thereof such asethyl-DHA. The composition further optionally comprises one or moreantioxidants (e.g. tocopherol) or other impurities in an amount of notmore than about 0.5% or not more than 0.05%. In another embodiment, thecomposition comprises about 0.05% to about 0.4%, for example about 0.2%by weight tocopherol. In another embodiment, about 500 mg to about 1 gof the composition is provided in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96% byweight ethyl eicosapentaenoate, about 0.22% to about 0.4% by weightethyl octadecatetraenoate, about 0.075% to about 0.20% by weight ethylnonaecapentaenoate, about 0.25% to about 0.40% by weight ethylarachidonate, about 0.3% to about 0.4% by weight ethyl eicosatetraenoateand about 0.075% to about 0.25% ethyl heneicosapentaenoate. Optionally,the composition contains not more than about 0.06%, about 0.05%, orabout 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. Inone embodiment the composition contains substantially no or no amount ofDHA or derivative thereof such as ethyl-DHA. The composition furtheroptionally comprises one or more antioxidants (e.g. tocopherol) or otherimpurities in an amount of not more than about 0.5% or not more than0.05%. In another embodiment, the composition comprises about 0.05% toabout 0.4%, for example about 0.2% by weight tocopherol. In anotherembodiment, the invention provides a dosage form comprising about 500 mgto about 1 g of the foregoing composition in a capsule shell. In oneembodiment, the dosage form is a gel or liquid capsule and is packagedin blister packages of about 1 to about 20 capsules per sheet.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96%,97% or 98%, by weight, ethyl eicosapentaenoate, about 0.25% to about0.38% by weight ethyl octadecatetraenoate, about 0.10% to about 0.15% byweight ethyl nonaecapentaenoate, about 0.25% to about 0.35% by weightethyl arachidonate, about 0.31% to about 0.38% by weight ethyleicosatetraenoate, and about 0.08% to about 0.20% ethylheneicosapentaenoate. Optionally, the composition contains not more thanabout 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivativethereof such as ethyl-DHA. In one embodiment the composition containssubstantially no or no amount of DHA or derivative thereof such asethyl-DHA. The composition further optionally comprises one or moreantioxidants (e.g. tocopherol) or other impurities in an amount of notmore than about 0.5% or not more than 0.05%. In another embodiment, thecomposition comprises about 0.05% to about 0.4%, for example about 0.2%by weight tocopherol. In another embodiment, the invention provides adosage form comprising about 500 mg to about 1 g of the foregoingcomposition in a capsule shell.

In another embodiment, a composition as described herein is administeredto a subject once or twice per day. In another embodiment, 1, 2, 3 or 4capsules, each containing about 1 g of a composition as describedherein, are administered to a subject daily. In another embodiment, 1 or2 capsules, each containing about 1 g of a composition as describedherein, are administered to the subject in the morning, for examplebetween about 5 am and about 11 am, and 1 or 2 capsules, each containingabout 1 g of a composition as described herein, are administered to thesubject in the evening, for example between about 5 pm and about 11 pm.

In one embodiment, a subject being treated in accordance with methods ofthe invention is not otherwise on lipid-altering therapy, for examplestatin, fibrate, niacin and/or ezetimibe therapy.

In another embodiment, compositions useful in accordance with methods ofthe invention are orally deliverable. The terms “orally deliverable” or“oral administration” herein include any form of delivery of atherapeutic agent or a composition thereof to a subject wherein theagent or composition is placed in the mouth of the subject, whether ornot the agent or composition is swallowed. Thus “oral administration”includes buccal and sublingual as well as esophageal administration. Inone embodiment, the composition is present in a capsule, for example asoft gelatin capsule.

A composition for use in accordance with the invention can be formulatedas one or more dosage units. The terms “dose unit” and “dosage unit”herein refer to a portion of a pharmaceutical composition that containsan amount of a therapeutic agent suitable for a single administration toprovide a therapeutic effect. Such dosage units may be administered oneto a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) oftimes per day, or as many times as needed to elicit a therapeuticresponse.

In another embodiment, the invention provides use of any compositiondescribed herein for treating moderate to severe hypertriglyceridemia ina subject in need thereof, comprising: providing a subject having afasting baseline triglyceride level of about 500 mg/dl to about 1500mg/dl and administering to the subject a pharmaceutical composition asdescribed herein. In one embodiment, the composition comprises about 1 gto about 4 g of eicosapentaenoic acid ethyl ester, wherein thecomposition contains substantially no docosahexaenoic acid.

In one embodiment, compositions of the invention, upon storage in aclosed container maintained at room temperature, refrigerated (e.g.about 5 to about 5-10° C.) temperature, or frozen for a period of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, exhibit at least about90%, at least about 95%, at least about 97.5%, or at least about 99% ofthe active ingredient(s) originally present therein.

In one embodiment, the invention provides use of a composition asdescribed herein in manufacture of a medicament for treatment of any ofa cardiovascular-related disease. In another embodiment, the subject isdiabetic.

In one embodiment, a composition as set forth herein is packagedtogether with instructions for using the composition to treat acardiovascular disorder.

EXAMPLES Example 1: Clinical Study to Evaluate TG Levels in SubjectsTreated with AMR101

A multi-center, placebo-controlled randomized, double-blind, 12-weekstudy with an open-label extension was performed to evaluate theefficacy and safety of AMR101 in patients with fasting triglyceridelevels ≥500 mg/dL. The primary objective of the study was to determinethe efficacy of AMR101 2 g daily and 4 g daily, compared to placebo, inlowering fasting TG levels in patients with fasting TG levels ≥500 mg/dLand ≤1500 mg/dL (≥5.65 mmol/L and ≤16.94 mmol/L).

The secondary objectives of this study were the following:

-   1. To determine the safety and tolerability of AMR101 2 g daily and    4 g daily;-   2. To determine the effect of AMR101 on lipid and apolipoprotein    profiles;-   3. To determine the effect of AMR101 on low-density lipoprotein    (LDL) particle number and size;-   4. To determine the effect of AMR101 on oxidized LDL;-   5. To determine the effect of AMR101 on fasting plasma glucose (FPG)    and hemoglobin A_(1c) (HbA_(1c));-   6. To determine the effect of AMR101 on insulin resistance;-   7. To determine the effect of AMR101 on high-sensitivity C-reactive    protein (hsCRP);-   8. To determine the effects of AMR101 2 g daily and 4 g daily on the    incorporation of fatty acids into red blood cell membranes and into    plasma phospholipids;-   9. To explore the relationship between baseline fasting TG levels    and the reduction in fasting TG levels; and-   10. To explore the relationship between an increase in red blood    cell membrane eicosapentaenoic acid (EPA) concentrations and the    reduction in fasting TG levels.

The population for this study wass men and women (women of childbearingpotential needed to be on contraception or practice abstinence) >18years of age with a body mass index ≤45 kg/m² who were not onlipid-altering therapy or are currently on lipid-altering therapy.Patients currently on statin therapy (with or without ezetimibe) wereevaluated by the investigator as to whether this therapy could be safelydiscontinued at screening, or if it should be continued. If statintherapy (with or without ezetimibe) was to be continued, dose(s) must bestable for ≥4 weeks prior to randomization. Patients taking non-statin,lipid-altering medications (niacin >200 mg/day, fibrates, fish oil,other products containing omega-3 fatty acids, or other herbal productsor dietary supplements with potential lipid-altering effects), eitheralone or in combination with statin therapy (with or without ezetimibe),must have been able to safely discontinue non-statin, lipid-alteringtherapy at screening.

Approximately 240 patients were sought to be randomized at approximately50 centers in North America, South America, Central America, Europe,India, and South Africa. The study was be a 58- to 60-week, Phase 3,multi-center study consisting of 3 study periods: (1) A 6- to 8-weekscreening period that included a diet and lifestyle stabilization andwashout period and a TG qualifying period; (2) A 12-week, double-blind,randomized, placebo-controlled treatment period; and (3) A 40-week,open-label, extension period.

During the screening period and double-blind treatment period, allvisits were to be within ±3 days of the scheduled time. During theopen-label extension period, all visits were to be within ±7 days of thescheduled time. The screening period included a 4- or 6-week diet andlifestyle stabilization period and washout period followed by a 2-weekTG qualifying period. Subjects must be stable for ≥weeks prior torandomization.

The screening visit (Visit 1) occurred for all patients at either 6weeks (for patients not on lipid-altering therapy at screening or forpatients who did not need to discontinue their current lipid-alteringtherapy) or 8 weeks (for patients who required washout of their currentlipid-altering therapy at screening) before randomization, as follows:

Patients who did not require a washout: The screening visit occurred atVisit 1 (Week −6). Eligible patients entered a 4-week diet and lifestylestabilization period. At the screening visit, all patients receivedcounseling regarding the importance of the National CholesterolEducation Program (NCEP) Therapeutic Lifestyle Changes (TLC) diet andreceived instructions on how to follow this diet. Patients who requireda washout: The screening visit occured at Visit 1 (Week −8). Eligiblepatients began a 6-week washout period at the screening visit. Patientsreceived counseling regarding the NCEP TLC diet and receivedinstructions on how to follow this diet. Site personnel contactedpatients who did not qualify for participation based on screeninglaboratory test results to instruct them to resume their priorlipid-altering medications.

At the end of the 4-week diet and lifestyle stabilization period or the6-week diet and stabilization and washout period, eligible patientsentered the 2-week TG qualifying period and had their fasting TG levelmeasured at Visit 2 (Week −2) and Visit 3 (Week −1). Eligible patientsmust have had an average fasting TG level ≥500 mg/dL and ≤1500 mg/dL(≥5.65 mmol/L and ≤16.94 mmol/L) to enter the 12-week double-blindtreatment period. The TG level for qualification was based on theaverage (arithmetic mean) of the Visit 2 (Week −2) and Visit 3 (Week −1)values. If a patient's average TG level from Visit 2 and Visit 3 felloutside the required range for entry into the study, an additionalsample for fasting TG measurement could be collected 1 week later atVisit 3.1. If a third sample was collected at Visit 3.1, entry into thestudy was based on the average (arithmetic mean) of the values fromVisit 3 and Visit 3.1.

After confirmation of qualifying fasting TG values, eligible patientsentered a 12-week, randomized, double-blind treatment period. At Visit 4(Week 0), patients were randomly assigned to 1 of the followingtreatment groups:

-   -   AMR101 2 g daily,    -   AMR101 4 g daily, or    -   Placebo.

During the double-blind treatment period, patients returned to the siteat Visit 5 (Week 4), Visit 6 (Week 11), and Visit 7 (Week 12) forefficacy and safety evaluations.

Patients who completed the 12-week double-blind treatment period wereeligible to enter a 40-week, open-label, extension period at Visit 7(Week 12). All patients were to receive open-label AMR101 4 g daily.From Visit 8 (Week 16) until the end of the study, changes to thelipid-altering regimen were permitted (e.g., initiating or raising thedose of statin or adding non-statin, lipid-altering medications to theregimen), as guided by standard practice and prescribing information.After Visit 8 (Week 16), patients were to return to the site every 12weeks until the last visit at Visit 11 (Week 52).

Eligible patients were randomly assigned at Visit 4 (Week 0) to receiveorally AMR101 2 g daily, AMR101 4 g daily, or placebo for the 12-weekdouble-blind treatment period. AMR101 was provided in 1 g liquid-filled,oblong, gelatin capsules. The matching placebo capsule was filled withlight liquid paraffin and contained 0 g of AMR101. During thedouble-blind treatment period, patients took 2 capsules (AMR101 ormatching placebo) in the morning and 2 in the evening for a total of 4capsules per day. Patients in the AMR101 2 g/day treatment groupreceived 1 AMR101 1 g capsule and 1 matching placebo capsule in themorning and in the evening. Patients in the AMR101 4 g/day treatmentgroup received 2 AMR101 1 g capsules in the morning and evening.

Patients in the placebo group received 2 matching placebo capsules inthe morning and evening. During the extension period, patients receivedopen-label AMR101 4 g daily. Patients took 2 AMR101 1 g capsules in themorning and 2 in the evening.

The primary efficacy variable for the double-blind treatment period waspercent change in TG from baseline to Week 12 endpoint. The secondaryefficacy variables for the double-blind treatment period included thefollowing:

-   -   Percent changes in total cholesterol (TC), high-density        lipoprotein cholesterol (HDL-C), calculated low-density        lipoprotein cholesterol (LDL-C), calculated non-high-density        lipoprotein cholesterol (non-HDL-C), and very low-density        lipoprotein cholesterol (VLDL-C) from baseline to Week 12        endpoint;    -   Percent change in very low-density lipoprotein TG from baseline        to Week 12;    -   Percent changes in apolipoprotein A-I (apo A-I), apolipoprotein        B (apo B), and apo A-I/apo B ratio from baseline to Week 12;    -   Percent changes in lipoprotein(a) from baseline to Week 12        (selected sites only);    -   Percent changes in LDL particle number and size, measured by        nuclear magnetic resonance, from baseline to Week 12 (selected        sites only);    -   Percent change in remnant-like particle cholesterol from        baseline to Week 12 (selected sites only);    -   Percent change in oxidized LDL from baseline to Week 12        (selected sites only);    -   Changes in FPG and HbA_(1c) from baseline to Week 12;    -   Change in insulin resistance, as assessed by the homeostasis        model index insulin resistance, from baseline to Week 12;    -   Percent change in lipoprotein associated phospholipase A2 from        baseline to Week 12 (selected sites only);    -   Change in intracellular adhesion molecule-1 from baseline to        Week 12 (selected sites only);    -   Change in interleukin-6 from baseline to Week 12 (selected sites        only);    -   Change in plasminogen activator inhibitor-1 from baseline to        Week 12 (selected sites only);    -   Change in hsCRP from baseline to Week 12 (selected sites only);    -   Change in serum phospholipid EPA content from baseline to Week        12;    -   Change in red blood cell membrane EPA content from baseline to        Week 12; and    -   Change in serum phospholipid and red blood cell membrane content        in the following fatty acids from baseline to Week 12:        docosapentaenoic acid, docosahexaenoic acid, arachidonic acid,        palmitic acid, stearic acid, and oleic acid.

The efficacy variable for the open-label extension period was percentchange in fasting TG from extension baseline to end of treatment. Safetyassessments included adverse events, clinical laboratory measurements(chemistry, hematology, and urinalysis), 12-lead electrocardiograms(ECGs), vital signs, and physical examinations

For TG, TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C,baseline was defined as the average of Visit 4 (Week 0) and thepreceding lipid qualifying visit (either Visit 3 [Week −1] or if itoccured, Visit 3.1) measurements. Baseline for all other efficacyparameters was the Visit 4 (Week 0) measurement.

For TC, HDL-C, calculated LDL-C, calculated non-HDL-C, and VLDL-C, Week12 endpoint was defined as the average of Visit 6 (Week 11) and Visit 7(Week 12) measurements. Week 12 endpoint for all other efficacyparameters was the Visit 7 (Week 12) measurement.

The primary efficacy analysis was performed using a 2-way analysis ofcovariance (ANCOVA) model with treatment as a factor and baseline TGvalue as a covariate. The least-squares mean, standard error, and2-tailed 95% confidence interval for each treatment group and for eachcomparison was estimated. The same 2-way ANCOVA model was used for theanalysis of secondary efficacy variables.

The primary analysis was repeated for the per-protocol population toconfirm the robustness of the results for the intent-to-treatpopulation.

The primary efficacy variable was the percent change in fasting TGlevels from baseline to Week 12. A sample size of 69 completed patientsper treatment group provided 90% power to detect a difference of 30%between AMR101 and placebo in percent change from baseline in fasting TGlevels, assuming a standard deviation of 45% in TG measurements and asignificance level of p<0.01. To accommodate a 15% drop-out rate fromrandomization to completion of the double-blind treatment period, atotal of 240 randomized patients was planned (80 patients per treatmentgroup). Top line results of the study are shown in Table 1.

TABLE 1 Lipid Parameter Results Median Baseline and Percent Change FromBaseline in Lipid Parameters in 224 Patients with Very High TG Levels(≥500 mg/dL) Median P-value Placebo AMR101 2 g/day AMR101 4 g/day AMR101AMR101 AMR101 AMR101 % % % 4 g/day vs. 2 g/day vs. 4 g/day vs. 2 g/dayvs. Parameter BL Change BL Change BL Change Placebo Placebo PlaceboPlacebo TG (mg/dL) 703.0 9.7 656.5 −7.0 679.5 −26.6 −33.1 −19.7 <0.00010.0051 VLDL-C (mg/dL) 124.0 13.7 119.0 0.0 122.5 −19.5 −28.6 −15.30.0002 0.0384 Lp-PLA₂ (ng/mL) 253.0 −2.4 235.0 −5.1 246.0 −17.1 −13.6−5.1 0.0003 0.1529 *Apo B (mg/dL) 118.0 4.3 117.5 2.1 121.0 −3.8 −8.5−2.6 0.0019 0.2367 TC (mg/dL) 256.0 7.7 236.0 0.7 253.5 −7.3 −16.3 −6.8<0.0001 0.0148 HDL-C (mg/dL) 27.0 0.0 26.0 0.0 26.5 −3.5 −3.6 1.5 0.21740.5225 LDL-C (mg/dL) 86.0 −3.0 84.0 −2.5 90.5 −4.5 −2.3 5.2 0.67680.3022 Non-HDL-C (mg/dL) 229.0 7.8 210.0 0.0 225.0 −7.7 −17.7 −8.1<0.0001 0.0182 *VLDL-TG (mg/dL) 543.0 7.8 488.0 −6.4 522.5 −25.2 −25.8−17.3 0.0023 0.0733 BL = Baseline (mg/dL); % Change = Median PercentChange from Baseline *Secondary endpoints which are pre-specified forinclusion in the Label and we may be able to call out in the Indicationsection

Example 2: Clinical Study to Evaluate Lipoprotein Particle Concentrationand Size in Subjects Treated with AMR101

Lipoprotein particle concentrations and sizes were measured with nuclearmagnetic resonance spectroscopy in the above described clinical trial.Lipoprotein particle size and concentration parameters were obtainedfrom fasting blood samples. Lipoprotein particle size and concentrationsare measured by nuclear magnetic resonance (NMR) spectroscopy atLipoScience, Inc. (Raleigh, N.C.) using the LipoProfile-3 algorithm.VLDL, LDL, and HDL subclasses of different size were quantified from theamplitudes of their spectroscopically-distinct lipid methyl group NMRsignals.

Concentrations of the following subclasses of lipoproteins were analyzedin this study: small LDL (18.0-20.5 nm), large LDL (20.5-23.0 nm),intermediate-density lipoprotein (IDL; 23.0-29.0 nm), large high-densitylipoprotein (HDL; 9.4-14.0 nm), medium HDL (8.2-9.4 nm), small HDL(7.3-8.2 nm), large very-low-density lipoprotein (VLDL; >60 nm), mediumVLDL (42-60 nm), and small VLDL (29-42 nm).

Efficacy analyses include the intent-to-treat (ITT) population, definedas all randomized patients who had a baseline efficacy measurement,received ≥1 dose of study drug, and had ≥1 postrandomization efficacymeasurement. Subjects with missing baseline or Week 12 measurements, dueto either missing laboratory samples or un-reportable values, wereexcluded from this analysis. The median difference of each lipoproteinparticle variable (percent change from baseline) between each AMR101treatment group and the placebo group was evaluated with a nonparametrictest using the Hodges-Lehmann medians of the differences betweentreatment groups and the Wilcoxon rank-sum test (significance level0.05). All statistical analyses were carried out using SAS 9.2 software.

AMR101 4 g/day significantly reduced the concentration of large VLDLparticles (−27.9%; P=0.0211) and increased the concentration of mediumVLDL particles (+28.0%, P=0.0238) with no significant change in medianconcentration of total VLDL particles (+9.3%, P=0.2262). AMR101 2 g/daydid not significantly change total, small, medium, or large VLDLparticle concentration. See, Table 2.

AMR101 4 g/day and 2 g/day both significantly reduced small LDL particleconcentration by 25.6% (P<0.0001) and 12.8% (P=0.0274), respectively,with no significant changes in concentrations of large LDL or IDLparticles. AMR101 4 g/day also reduced total LDL particle concentration(which included IDL particles, small LDL particles, and large LDLparticles) by 16.3% (P=0.0006). AMR 101 2 g/day did not significantlychange total LDL particle concentration (−1.1%, P=0.8202). See, Table 2.

AMR101 4 g/day significantly reduced total HDL particle concentration(−7.4%, P=0.0063); AMR101 2 g/day did not (−3.0%, P=0.2701). Neitherdose of AMR101 significantly changed concentrations of large, medium, orsmall HDL particles. See, Table 2.

AMR101 4 g/day was previously reported to significantly reduce medianapolipoprotein B by 8.5% compared with placebo (P=0.0019), which wasconsistent with the reductions in the atherogenic particleconcentrations observed in the present analysis. The concentration ofall LDL and VLDL particles correlated with apolipoprotein Bconcentration at week 12 (r=0.79, P<0.0001; see, FIG. 1).

TABLE 2 Median percent change from baseline to study end in lipoproteinparticle concentrations Placebo-adjusted median percent change AMR101 4g/day (n = 61) AMR101 2 g/day (n = 63) Placebo (n = 53) from baselineEnd-of- Change End-of- Change End-of- Change AMR101 AMR101 Baselinetreatment from Baseline treatment from Baseline treatment from 4 g/dayvs 2 g/day vs value value baseline, value value baseline, value valuebaseline, placebo, placebo, (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR)(IQR) (IQR) % (IQR) %, P %, P Total VLDL 166.2 200.3 15.2 165.5 190.113.1 161.8 169.1 1.2 9.3 5.4 (85.2) (150.5) (58.6) (88.9) (121.3) (55.4)(71.6) (87.0) (37.1) 0.2262 0.4644 Large VLDL 33.3 21.2 −24.5 33.2 29.9−2.5 33.1 35.6 2.5 −27.9 −8.4 (26.8) (27.9) (75.5) (31.2) (29.2) (69.0)(23.5) (34.4) (80.7) 0.0211 0.4313 Medium VLDL 80.6 106.8 36.4 76.9 93.818.8 74.5 86.0 6.3 28.0 9.3 (68.6) (95.2) (89.7) (52.2) (89.2) (69.8)(55.0) (40.3) (72.1) 0.0238 0.4152 Small VLDL 56.1 52.5 4.3 41.3 53.911.5 48.8 45.5 −10.5 5.3 23.1 (30.6) (55.1) (119.1) (48.8) (49.8)(123.9) (35.3) (50.7) (74.5) 0.7253 0.1007 Total LDL 1418 1419 −0.1 13741464 12.6 1310 1452 14.4 −16.3 −1.1 (544.0) (718.0) (27.5) (556.0)(810.0) (28.7) (462.0) (679.0) (31.7) 0.0006 0.8202 IDL 160.0 185.0 19.2121.0 144.0 0.8 174.0 154.0 −14.2 26.6 25.3 (130.0) (217.0) (120.2)(153.0) (201.0) (190.8) (239.0) (161.0) (92.6) 0.1156 0.1111 Large LDL7.0 64.0 −32.3 9.0 51.0 −20.9 3.0 21.0 −22.9 0.0 5.3 (65.0) (174.0)(287.1) (116.0) (181.0) (151.0) (73.0) (158.0) (218.4) 0.8057 0.6996Small LDL 1132 998.0 −9.7 1155 1100 9.5 1001 1171 14.6 −25.6 −12.8(486.0) (568.0) (33.8) (481.0) (724.0) (29.8) (476.0) (524.0) (38.6)<0.0001 0.0274 Total HDL 34.3 30.8 −7.1 34.7 33.4 −3.3 32.9 33.9 −0.7−7.4 −3.0 (8.7) (9.1) (17.2) (8.9) (7.1) (19.6) (10.0) (9.1) (12.7)0.0063 0.2701 Large HDL 2.2 2.3 8.3 2.1 2.3 −1.6 2.2 2.5 0.0 8.5 2.0(1.4) (1.3) (67.0) (1.9) (2.0) (85.6) (1.6) (3.0) (89.3) 0.4527 0.8377Medium HDL 10.8 8.4 −18.3 10.0 8.7 3.9 10.8 10.1 −12.6 −14.9 −4.2 (9.2)(5.3) (81.6) (7.9) (7.3) (68.7) (8.3) (9.3) (89.7) 0.1223 0.6415 SmallHDL 20.6 21.6 −5.1 22.1 21.5 −3.2 18.8 17.9 −1.7 11.8 3.4 (7.2) (8.9)(34.6) (8.1) (6.7) (34.4) (6.6) (10.9) (37.1) 10.8247 0.5756Concentrations are expressed as nmol/L except for HDL and HDLsubtractions, which are expressed as μmol/L. Data are presented asmedian (IQR) for end point values. Placebo-adjusted median percentchanges are Hodges-Lehmann medians. HDL = high-density lipoprotein; IDL= intermediate-density lipoprotein; IQR = interquartile range; LDL =low-density lipoprotein; VLDL = very-low-density lipoprotein.

At study end and compared with placebo, AMR101 4 g/day significantlydecreased VLDL particle size (−8.6%, P=0.0017). AMR101 2 g/day alsodecreased VLDL particle size, although not significantly so (−4.0%,P=0.0734). Neither AMR101 dose significantly changed LDL or HDL particlesize. See, Table 3.

TABLE 3 Median percent change from baseline to study end in lipoproteinparticle size (nm) Placebo-adjusted median percent change AMR101 4 g/day(n = 61*) AMR101 2 g/day (n = 63) Placebo (n = 53) from baseline End-of-Change End-of- Change End-of- Change AMR101 AMR101 Baseline treatmentfrom Baseline treatment from Baseline treatment from 4 g/day vs 2 g/dayvs value value baseline, value value baseline, value value baseline,placebo, placebo, (IQR) (IQR) % (IQR) (IQR) (IQR) % (IQR) (IQR) (IQR) %(IQR) %, P %, P VLDL 62.3 55.7 −8.6 64.9 60.6 −3.4 66.8 65.7 −1.05 −8.6−4.0 (12.3) (13.1) (19.6) (12.3) (16.4) (14.9) (15.7) (15.0) (12.1)0.0017 0.0734 LDL 19.6 19.7 0.0 19.6 19.6 0.0 19.6 19.6 0.0 0.5 0.0(0.2) (0.6) (2.6) (0.3) (0.5) (2.1) (0.2) (0.3) (1.0) 0.4219 0.8695 HDL8.9 8.9 1.1 8.8 8.9 0.0 8.9 8.9 0.0 1.1 0.0 (0.4) (0.4) (4.3) (0.4)(0.4) (3.4) (0.4) (0.4) (3.3) 0.1152 0.7390 *n = 60 for LDL. Data arepresented as median (interquartile range) for end point values.Placebo-adjusted median percent changes are Hodges-Lehmann medians. HDL= high-density lipoprotein; IQR = interquartile range; LDL = low-densitylipoprotein; VLDL = very low-density lipoprotein.

1-9. (canceled)
 10. A method of reducing small LDL particleconcentration in a subject having a fasting baseline triglyceride levelof about 500 mg/dl to about 1500 mg/dl and a baseline small LDL particleconcentration of about 800 nmol/L to about 1400 nmol/L, comprising,administering to the subject about 4 g per day of ethyleicosapentaenoate thereby to produce a reduction in small LDL particleconcentration by comparison with a baseline small LDL particleconcentration prior to initiation of administration, wherein the smallLDL particle has a diameter of 18.0-20.5 nm.
 11. The method of claim 10wherein the reduction in small LDL particle concentration is at least a5% reduction.
 12. The method of claim 10 wherein the reduction in smallLDL particle concentration is at least a 9% reduction.
 13. The method ofclaim 10 wherein the subject is administered about 3.8 g to about 4 gper day of ethyl eicosapentaenoate.
 14. The method of claim 10 whereinthe subject is administered ethyl eicosapentaenoate for a period of 12weeks.